PROJECT SUMMARY Fecal Microbiota Transplantation (FMT) has emerged as a promising therapy for microbiome-related diseases following its success as a definitive treatment of recurring Clostridium difficile infections (CDI). However, initial placebo-controlled clinical trials of FMT for the treatment of Ulcerative Colitis have failed to show similar rates of clinical efficacy. A review of several FMT trials reveals that they commonly differ in how FMT recipients are prepared for transplant, the frequency and dosage of FMT administration, and the selection criteria for recipients and donors. The limited success of FMT for other diseases may be in part due to this methodological variability between clinical trials. This proposal aims to improve our understanding of the factors that contribute to successful FMTs, with the goal of identifying key parameters of FMT that can be altered to improve engraftment and efficacy for diseases other than CDI. To test several parameters that may contribute to FMT success in a well-controlled manner, we will utilize a novel gnotobiotic murine model of FMT and measure efficacy with a standardized set of FMT-specific quantitative metrics we have developed. This experimental approach allows us to investigate how the human gut microbiota responds to perturbations with better control over environmental variables that make similar studies in humans difficult to interpret. Aim 1 ? Based on our preliminary findings, we anticipate that recipient pre-treatment with antibiotics or other microbiome-altering agents can improve donor microbiota engraftment and recipient microbiota removal. We aim to identify the effect of recipient pre-treatment and FMT dosage on transplant success by testing several pharmacologic interventions and FMT schedules in our murine model. These data will allow us to develop an optimized FMT protocol that can be readily adopted by clinical FMT trials. Furthermore, in Aim 2, we will evaluate the effect of active colonic inflammation on FMT engraftment and efficacy. We will employ the T cell transfer mouse colitis model to study whether active inflammation influences FMT success, and whether FMT can alter the disease course. These experiments will allow us to determine whether host inflammation presents a significant challenge for the application of FMT to diseases such as Inflammatory Bowel Disease. By studying which factors affect FMT engraftment and efficacy, we can develop optimized protocols that can be utilized in clinical settings to expand the use of FMT beyond CDI.